Histone deacetylase inhibitors

ABSTRACT

Histone deacetylase is a metallo-enzyme with zinc at the active site. Compounds having a zinc-binding moiety, such as, for example, a hydroxamic acid group or a carboxylic acid group, can inhibit histone deacetylase. Histone deacetylase can repress gene expression, including expression of genes related to tumor suppression. Accordingly, inhibition of histone deacetylase can provide an alternate route for treating cancer, hematological disorders, e.g., hemoglobinopathies, and genetic related metabolic disorders, e.g., cystic fibrosis and adrenoleukodystrophy.

This application is a divisional of U.S. application Ser. No.09/812,945, filed Mar. 27, 2001 now U.S. Pat. No. 7,312,247, thedisclosure of which is incorporated by reference herein.

TECHNICAL FIELD

This invention relates to enzyme inhibitors, and more particularly tohistone deacetylase inhibitors.

BACKGROUND

DNA in the nucleus of the cell exists as a hierarchy of compactedchromatin structures. The basic repeating unit in chromatin is thenucleosome. The nucleosome consists of a histone octomer of proteins inthe nucleus of the cell around which DNA is twice wrapped. The orderlypackaging of DNA in the nucleus plays an important role in thefunctional aspects of gene regulation. Covalent modifications of thehistones have a key role in altering chromatin higher order structureand function and ultimately gene expression. The covalent modificationof histones occurs by enzymatically mediated processes, such asacetylation.

Regulation of gene expression through the inhibition of the nuclearenzyme histone deacetylase (HDAC) is one of several possible regulatorymechanisms whereby chromatin activity can be affected. The dynamichomeostasis of the nuclear acetylation of histones can be regulated bythe opposing activity of the enzymes histone acetyl transferase (HAT)and histone deacetylase (HDAC). Transcriptionally silent chromatin canbe characterized by nucleosomes with low levels of acetylated histones.Acetylation of histones reduces its positive charge, thereby expandingthe structure of the nucleosome and facilitating the interaction oftranscription factors to the DNA. Removal of the acetyl group restoresthe positive charge condensing the structure of the nucleosome.Acetylation of histone-DNA activates transcription of DNA's message, anenhancement of gene expression. Histone deacetylase can reverse theprocess and can serve to repress gene expression. See, for example,Grunstein, Nature 389, 349-352 (1997); Pazin et al., Cell 89, 325-328(1997); Wade et al., Trends Biochem. Sci. 22, 128-132 (1997); andWolffe, Science 272, 371-372 (1996).

SUMMARY

Histone deacetylase is a metallo-enzyme with zinc at the active site.Compounds having a zinc-binding moiety, such as, for example, ahydroxamic acid group or a carboxylic acid group, can inhibit histonedeacetylase. Histone deacetylase can repress gene expression, includingexpression of genes related to tumor suppression. Accordingly,inhibition of histone deacetylase can provide an alternate route fortreating cancer, hematological disorders, e.g., hemoglobinopathies, andgenetic related metabolic disorders, e.g., cystic fibrosis andadrenoleukodystrophy.

In one aspect, a method of inhibiting histone deacetylation activity incells includes contacting the cells with an effective amount of acompound of formula (I), thereby treating one or more disorders mediatedby histone deacetylase, and determining whether the level of acetylatedhistones in the treated cells is higher than in untreated cells underthe same conditions.

The compound of formula (I) can be the following:

A is a cyclic moiety selected from the group consisting of C₃₋₁₄cycloalkyl, 3-14 membered heterocycloalkyl, C₄₋₁₄ cycloalkenyl, 4-14membered heterocycloalkenyl, aryl, or heteroaryl; the cyclic moietybeing optionally substituted with alkyl, alkenyl, alkynyl, alkoxy,hydroxyl, hydroxylalkyl, halo, haloalkyl, amino, alkylcarbonyloxy,alkyloxycarbonyl, alkylcarbonyl, alkylsulfonylamino, aminosulfonyl, oralkylsulfonyl; or A is a saturated branched C₃₋₁₂ hydrocarbon chain oran unsaturated branched C₃₋₁₂ hydrocarbon chain optionally interruptedby —O—, —S—, —N(R^(a))—, —C(O)—, —N(R^(a))—SO₂—, —SO₂—N(R^(a))—,—N(R^(a))—C(O)—O—, —O—C(O)—N(R^(a))—, —N(R^(a))—C(O)—N(R^(b))—,—O—C(O)—, —C(O)—O—, —O—SO₂—, —SO₂—O—, or —O—C(O)—O—, where each of R^(a)and R^(b), independently, is hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, or haloalkyl; each of the saturated and theunsaturated branched hydrocarbon chain being optionally substituted withalkyl, alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo,haloalkyl, amino, alkylcarbonyloxy, alkyloxycarbonyl, alkylcarbonyl,alkylsulfonylamino, aminosulfonyl, or alkylsulfonyl. Each of Y¹ and Y²,independently, is —CH₂—, —O—, —S—, —N(R^(c))—, —N(R^(c))—C(O)—O—,—O—C(O)—N(R^(c))—, —N(R^(c))—C(O)—N(R^(d))—, —O—C(O)—O—, or a bond, andeach of R^(c) and R^(d), independently, being hydrogen, alkyl, alkenyl,alkynyl, alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. L is a straightC₂₋₁₂ hydrocarbon chain optionally containing at least one double bond,at least one triple bond, or at least one double bond and one triplebond; said hydrocarbon chain being optionally substituted with C₁₋₄alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, hydroxyl, halo, amino,nitro, cyano, C₃₋₅ cycloalkyl, 3-5 membered heterocycloalkyl, monocyclicaryl, 5-6 membered heteroaryl, C₁₋₄ alkylcarbonyloxy, C₁₋₄alkyloxycarbonyl, C₁₋₄ alkylcarbonyl, or formyl; and further beingoptionally interrupted by —O—, —N(R^(e))—, —N(R^(e))—C(O)—O—,—O—C(O)—N(R^(e))—, —N(R^(e))—C(O)—N(R^(f))—, or —O—C(O)—O—; each ofR^(e) and R^(f), independently, being hydrogen, alkyl, alkenyl, alkynyl,alkoxy, hydroxylalkyl, hydroxyl, or haloalkyl. X¹ is O or S. X² is —OR¹,—SR¹, —NR³—OR, —NR³—SR¹, —C(O)—OR¹, —CHR¹—OR¹, —N═N—C(O)—N(R³)₂, or—O—CHR⁴—O—C(O)—R⁵, where each of R¹ and R², independently, is hydrogen,alkyl, hydroxylalkyl, haloalkyl, or a hydroxyl protecting group, R³ ishydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl,haloalkyl, or an amino protecting group, R⁴ is hydrogen, alkyl,hydroxylalkyl, or haloalkyl, and R⁵ is alkyl, hydroxylalkyl, orhaloalkyl. When L is a C₂₋₃ hydrocarbon containing no double bonds andX² is —OR¹, Y¹ is not a bond and Y² is not a bond.

In another aspect, a method of inhibiting histone deacetylase in cellscomprising contacting the cells with an effective amount of a compoundof formula (I), supra, and determining whether the level of acetylatedhistones in the treated cells is higher than in untreated cells underthe same conditions. A is phenyl optionally substituted with alkyl,alkenyl, alkynyl, alkoxy, hydroxyl, hydroxylalkyl, halo, haloalkyl, oramino. Each of Y¹ and Y², independently, is —CH₂—, —O—, —S—, —N(R^(c))—,or a bond; where R^(c) is hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, or haloalkyl. L is a straight C₂₋₁₂ hydrocarbonchain optionally containing at least one double bond, at least onetriple bond, or at least one double bond and one triple bond, thehydrocarbon chain being optionally substituted with C₁₋₄ alkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, hydroxyl, halo, amino, nitro, cyano,C₃₋₅ cycloalkyl, 3-5 membered heterocycloalkyl, monocyclic aryl, 5-6membered heteroaryl, C₁₋₄ alkylcarbonyloxy, C₁₋₄ alkyloxycarbonyl, C₁₋₄alkylcarbonyl, or formyl; and further being optionally interrupted by—O—, —N(R^(e))—, —N(R^(e))—C(O)—O—, —O—C(O)—N(R^(e))—,—N(R^(e))—C(O)—N(R^(f))—, or —O—C(O)—O—, and each of R^(e) and R^(f),independently, being hydrogen, alkyl, alkenyl, alkynyl, alkoxy,hydroxylalkyl, hydroxyl, or haloalkyl. X¹ is O or S. X² is —OR¹, —SR¹,—NR³—OR¹, —NR³—SR¹, —C(O)—OR¹, —CHR⁴—OR¹, —N═N—C(O)—N(R³)₂, or—O—CHR⁴—O—C(O)—R⁵, where each of R¹ and R², independently, is hydrogen,alkyl, hydroxylalkyl, haloalkyl, or a hydroxyl protecting group, R³ ishydrogen, alkyl, alkenyl, alkynyl, alkoxy, hydroxylalkyl, hydroxyl,haloalkyl, or an amino protecting group, R⁴ is hydrogen, alkyl,hydroxylalkyl, or haloalkyl, and R⁵ is alkyl, hydroxylalkyl, orhaloalkyl. When L is a C₂₋₃ hydrocarbon containing no double bonds andX² is —OR¹, Y¹ is not a bond and Y² is not a bond.

In yet another aspect, a method of treating a histonedeacetylase-mediated disorder includes administering to a subject inneed thereof a therapeutically effective amount of compound of formula(I), supra.

In certain embodiments, X² can be —OR¹, —NR³—OR¹, —C(O)—OR¹, —CHR¹—OR¹,or —O—CHR⁴—O—C(O)—R⁵, X² can be —OR¹, —NR³—OR¹, —C(O)OR¹, or—O—CHR⁴—O—C(O)—R⁵, each of Y¹ and Y², independently, can be —CH₂—, —O—,—N(R^(c))—, or a bond, L can be a saturated hydrocarbon chain, such as aC₃₋₈ hydrocarbon chain substituted with C₁₋₂ alkyl, C₁₋₂ alkoxy,hydroxyl, —NH₂, —NH(C₁₋₂ alkyl), or —N(C₁₋₂ alkyl)₂.

In other embodiments, L can be an unsaturated hydrocarbon chaincontaining at least one double bond and no triple bond, such as anunsaturated C₄₋₈ hydrocarbon chain substituted with C₁₋₂ alkyl, C₁₋₂alkoxy, hydroxyl, —NH₂, —NH(C₁₋₂ alkyl), or —N(C₁₋₂ alkyl)₂. In otherembodiments, L can be an unsaturated hydrocarbon chain containing atleast one double bond and one triple bond, such as an unsaturated C₄₋₈hydrocarbon chain substituted with C₁₋₂ alkyl, C₁₋₂ alkoxy, hydroxyl,—NH₂, —NH(C₁₋₂ alkyl), or —N(C₁₋₂ alkyl)₂. The double bond can be intrans configuration.

In certain embodiments, A can be a C₅₋₈ cycloalkenyl or 5-8 memberedheteroalkenyl containing at least one double bond. A can be phenyl,naphthyl, indanyl, or tetrahydronaphthyl, or A can be phenyl optionallysubstituted with alkyl alkenyl, alkynyl, alkoxy, hydroxyl,hydroxylalkyl, halo, haloalkyl, or amino. In other embodiments, A cancontain only double bonds.

Set forth below are some examples of a compound that can be employed inthe methods of the present invention: 5-phenyl-2,4-pentadienoic acid,3-methyl-5-phenyl-2,4-pentadienoic acid,4-methyl-5-phenyl-2,4-pentadienoic acid,4-chloro-5-phenyl-2,4-pentadienoic acid,5-(4-dimethylaminophenyl)-2,4-pentadienoic acid,5-(2-furyl)-2,4-pentadienoic acid, 5-phenyl-2-en-4-yn-pentanoic acid,6-phenyl-3,5-hexadienoic acid, 7-phenyl-2,4,6-heptatrienoic acid,8-phenyl-3,5,7-octatrienoic acid, potassium2-oxo-6-phenyl-3,5-hexadienoate, potassium2-oxo-8-phenyl-3,5,7-octatrienoate, cinnamoylhydroxamic acid,methyl-cinnamoylhydroxamic acid, 4-cyclohexanebutyroylhydroxamic acid,benzylthioglycoloylhydroxamic acid, 5-phenylpentanoylhydroxamic acid,5-phenyl-2,4-pentadienoylhydroxamic acid,N-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid,3-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid,4-methyl-5-phenyl-2,4-pentadienoyl hydroxamic acid,4-chloro-5-phenyl-2,4-pentadienoylhydroxamic acid,5-(4-dimethylaminophenyl)-2,4-pentadienoylhydroxamic acid,5-phenyl-2-en-4-yn-pentanoylhydroxamic acid,5-(2-furyl)-2,4-pentadienoylhydroxamic acid, 6-phenylhexanoylhydroxamicacid, 6-phenyl-3,5-hexadienoylhydroxamic acid,N-methyl-6-phenyl-3,5-hexadienoylhydroxamic acid,7-phenylheptanoylhydroxamic acid,7-phenyl-2,4,6-hepta-trienoylhydroxamic acid or8-phenyloctanoylhydroxamic acid.

A salt of any of the compounds of the invention can be prepared. Forexample, a pharmaceutically acceptable salt can be formed when anamino-containing compound of this invention reacts with an inorganic ororganic acid. Some examples of such an acid include hydrochloric acid,hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid,p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid,benzoic acid, and acetic acid. Examples of pharmaceutically acceptablesalts thus formed include sulfate, pyrosulfate bisulfate, sulfite,bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate,sebacate, fumarate, and maleate. A compound of this invention may alsoform a pharmaceutically acceptable salt when a compound of thisinvention having an acid moiety reacts with an inorganic or organicbase. Such salts include those derived from inorganic or organic bases,e.g., alkali metal salts such as sodium, potassium, or lithium salts;alkaline earth metal salts such as calcium or magnesium salts; orammonium salts or salts of organic bases such as morpholine, piperidine,pyridine, dimethylamine, or diethylamine salts.

It should be recognized that a compound of the invention can containchiral carbon atoms. In other words, it may have optical isomers ordiastereoisomers.

Alkyl is a straight or branched hydrocarbon chain containing 1 to 10(preferably, 1 to 6; more preferably 1 to 4) carbon atoms. Examples ofalkyl include, but are not limited to, methyl, ethyl, propyl, isopropyl,butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylhexyl, and3-ethyloctyl.

The terms “alkenyl” and “alkynyl” refer to a straight or branchedhydrocarbon chain containing 2 to 10 carbon atoms and one or more(preferably, 1-4 or more preferably 1-2) double or triple bonds,respectively. Some examples of alkenyl and alkynyl are allyl, 2-butenyl,2-pentenyl, 2-hexenyl, 2-butynyl, 2-pentynyl, and 2-hexynyl.

Cycloalkyl is a monocyclic, bicyclic or tricyclic alkyl group containing3 to 14 carbon atoms. Some examples of cycloalkyl are cyclopropyl,cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, and norbornyl.Heterocycloalkyl is a cycloalkyl group containing at least oneheteroatom (e.g., 1-3) such as nitrogen, oxygen, or sulfur. The nitrogenor sulfur may optionally be oxidized and the nitrogen may optionally bequaternized. Examples of heterocycloalkyl include piperidinyl,piperazinyl, tetrahydropyranyl, tetrahydrofuryl, and morpholinyl.Cycloalkenyl is a cycloalkyl group containing at least one (e.g., 1-3)double bond. Examples of such a group include cyclopentenyl,1,4-cyclohexa-di-enyl, cycloheptenyl, and cyclooctenyl groups. By thesame token, heterocycloalkenyl is a cycloalkenyl group containing atleast one heteroatom selected from the group of oxygen, nitrogen orsulfur.

Aryl is an aromatic group containing a 5-14 ring and can contain fusedrings, which may be saturated, unsaturated, or aromatic. Examples of anaryl group include phenyl, naphthyl, biphenyl, phenanthryl, andanthracyl. If the aryl is specified as “monocyclic aryl,” if refers toan aromatic group containing only a single ring, i.e., not a fused ring.

Heteroaryl is aryl containing at least one (e.g., 1-3) heteroatom suchas nitrogen, oxygen, or sulfur and can contain fused rings. Someexamples of heteroaryl are pyridyl, furanyl, pyrrolyl, thienyl,thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, andbenzthiazolyl.

The cyclic moiety can be a fused ring formed from two or more of thejust-mentioned groups. Examples of a cyclic moiety having fused ringsinclude fluorenyl, dihydro-dibenzoazepine, dibenzocycloheptenyl,7H-pyrazino[2,3-c]carbazole, or 9,10-dihydro-9,10-[2]buteno-anthracene.

Amino protecting groups and hydroxy protecting groups are well-known tothose in the art. In general, the species of protecting group is notcritical, provided that it is stable to the conditions of any subsequentreaction(s) on other positions of the compound and can be removedwithout adversely affecting the remainder of the molecule. In addition,a protecting group may be substituted for another after substantivesynthetic transformations are complete. Examples of an amino protectinggroup include, but not limited to, carbamates such as2,2,2-trichloroethylcarbamate or tertbutylcarbamate. Examples of ahydroxyl protecting group include, but not limited to, ethers such asmethyl, t-butyl, benzyl, p-methoxybenzyl, p-nitrobenzyl, allyl, trityl,methoxymethyl, 2-methoxypropyl, methoxyethoxymethyl, ethoxyethyl,tetrahydropyranyl, tetrahydrothiopyranyl, and trialkylsilyl ethers suchas trimethylsilyl ether, triethylsilyl ether, dimethylarylsilyl ether,triisopropylsilyl ether and t-butyldimethylsilyl ether; esters such asbenzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetylsuch as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl;and carbonates including but not limited to alkyl carbonates having fromone to six carbon atoms such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl; isobutyl, and n-pentyl; alkyl carbonates having fromone to six carbon atoms and substituted with one or more halogen atomssuch as 2,2,2-trichloroethoxymethyl and 2,2,2-trichloro-ethyl; alkenylcarbonates having from two to six carbon atoms such as vinyl and allyl;cycloalkyl carbonates having from three to six carbon atoms such ascyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl; and phenyl orbenzyl carbonates optionally substituted on the ring with one or moreC₁₋₆ alkoxy, or nitro. Other protecting groups and reaction conditionscan be found in T. W. Greene, Protective Groups in Organic Synthesis,(3rd, 1999, John Wiley & Sons, New York, N.Y.).

Note that an amino group can be unsubstituted (i.e., —NH₂),mono-substituted (i.e., —NHR), or di-substituted (i.e., —NR₂). It can besubstituted with groups (R) such as alkyl, cycloalkyl, heterocycloalkyl,aryl, heteroaryl, aralkyl, or heteroaralkyl. Halo refers to fluoro,chloro, bromo, or iodo.

Inhibition of a histone deacetylase in a cell is determined by measuringthe level of acetylated histones in the treated cells and measuring thelevel of acetylated histones in untreated cells and comparing thelevels. If the level of histone acetylation in the treated cellsincreases relative to the untreated cells, histone deacetylase has beeninhibited.

Some disorders or physiological conditions may be mediated byhyperactive histone deacetylase activity. A disorder or physiologicalcondition that is mediated by histone deacetylase refers to a disorderor condition wherein histone deacetylase plays a role in triggering theonset thereof. Examples of such disorders or conditions include, but notlimited to, cancer, hemoglobinopathies (e.g., thalassemia or sickle cellanemia), cystic fibrosis, protozoan infection, adrenoleukodystrophy,alpha-1 anti-trypsin, retrovirus gene vector reactivation, woundhealing, hair growth, peroxisome biogenesis disorder, andadrenoleukodystrophy.

Other features or advantages will be apparent from the followingdetailed description of several embodiments, and also from the appendedclaims.

DETAILED DESCRIPTION

A carboxylic acid-containing compound of the present invention can beprepared by any known methods in the art. For example, a compound of theinvention having an unsaturated hydrocarbon chain between A and —C(═X¹)—can be prepared according to the following scheme:

where L′ is a saturated or unsaturated hydrocarbon linker between A and—CH═CH— in a compound of the invention, and A and X¹ has the samemeaning as defined above. See Coutrot et al., Syn. Comm. 133-134 (1978).Briefly, butyllithium was added to an appropriate amount of anhydroustetrahydrofuran (THF) at a very low temperature (e.g., −65° C.). Asecond solution having diethylphosphonoacetic acid in anhydrous THF wasadded dropwise to the stirred butyllithium solution at the same lowtemperature. The resulting solution is stirred at the same temperaturefor an additional 30-45 minutes which is followed by the addition of asolution containing an aromatic acrylaldehyde in anhydrous THF over 1-2hours. The reaction mixture is then warmed to room temperature andstirred overnight. It is then acidified (e.g., with HCl) which allowsthe organic phase to be separated. The organic phase is then dried,concentrated, and purified (e.g., by recrystallization) to form anunsaturated carboxylic acid.

Alternatively, a carboxylic acid-containing compound can be prepared byreacting an acid ester of the formula A-L′-C(═O)—O-lower alkyl with aGrignard reagent (e.g., methyl magnesium iodide) and a phosphorusoxychloride to form a corresponding aldehyde, which can be furtheroxidized (e.g., by reacting with silver nitrate and aqueous NaOH) toform an unsaturated carboxylic acid.

Other types of carboxylic acid-containing compounds (e.g., thosecontaining a linker with multiple double bonds or triple bonds) can beprepared according to published procedures such as those described, forexample, in Parameswara et al., Synthesis, 815-818 (1980) and Denny etal., J. Org. Chem., 27, 3404 (1962).

Carboxylic acid-containing compounds described above can then beconverted to hydroxamic acid-containing compounds according to thefollowing scheme:

Triethylamine (TEA) is added to a cooled (e.g., 0-5° C.) anhydrous THFsolution containing the carboxylic acid. Isobutyl chloroformate is thenadded to the solution having carboxylic acid, which is followed by theaddition of hydroxylamine hydrochloride and TEA. After acidification,the solution was filtered to collect the desired hydroxamic acid.

An N-substituted hydroxamic acid can be prepared in a similar manner asdescribed above. A corresponding carboxylic acid A-L′-C(═O)—OH can beconverted to an acid chloride by reacting with oxalyl chloride (inappropriate solvents such as methylene chloride and dimethylformamide),which in turn, can be converted to a desired N-substituted hydroxamicacid by reacting the acid chloride with an N-substituted hydroxylaminehydrochloride (e.g., CH₃NHOH.HCl) in an alkaline medium (e.g., 40% NaOH(aq)) at a low temperature (e.g., 0-5° C.). The desired N-substitutedhydroxamic acid can be collected after acidifying the reaction mixtureafter the reaction has completed (e.g., in 2-3 hours).

As to compounds of the invention wherein X¹ is S, they can be preparedaccording to procedures described in Sandier, S. R. and Karo, W.,Organic Functional Group Preparations, Volume III (Academic Press, 1972)at pages 436-437. For preparation of compounds of the invention whereinX² is —N(R^(c))OH— and X¹ is S, see procedures described in U.S. Pat.Nos. 5,112,846; 5,075,330 and 4,981,865.

Compounds of the invention containing an α-keto acid moiety (e.g., whenX¹ is oxygen and X² is —C(═O)—OM or A-L′-C(═O)—C(═O)—OM, where A and L′have been defined above and M can be hydrogen, lower alkyl or a cationsuch as K⁺), these compounds can be prepared by procedures based on thatdescribed in Schummer et al., Tetrahedron, 43, 9019 (1991). Briefly, theprocedure starts with a corresponding aldehyde-containing compound(e.g., A-L′-C(═O)—H), which is allowed to react with a pyruvic acid in abasic condition (KOH/methanol) at a low temperature (e.g., 0-5° C.).Desired products (in the form of a potassium salt) are formed uponwarming of the reaction mixture to room temperature.

The compounds described above, as well as their (thio)hydroxamic acid orα-keto acid counterparts, can possess histone deacetylase inhibitoryproperties.

Note that appropriate protecting groups may be needed to avoid formingside products during the preparation of a compound of the invention. Forexample, if the linker L′ contains an amino substituent, it can be firstprotected by a suitable amino protecting group such as trifluoroacetylor tert-butoxycarbonyl prior to being treated with reagents such asbutyllithium. See, e.g., T. W. Greene, supra, for other suitableprotecting groups.

A compound produced by the methods shown above can be purified by flashcolumn chromatography, preparative high performance liquidchromatography, or crystallization.

A pharmaceutical composition can be used to inhibit histone deacetylasein cells and can be used to treat disorders associated with abnormalhistone deacetylase activity. Some examples of these disorders arecancers (e.g., leukemia, lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, cervical cancer, renal cancer, prostatecancer, and breast cancer), hematological disorders (e.g.,hemoglobinopathies, thalassemia, and sickle cell anemia) and geneticrelated metabolic disorders (e.g., cystic fibrosis, peroxisomebiogenesis disorder, alpha-1 anti-trypsin, and adrenoleukodystrophy).The compounds of this invention can also stimulate hematopoietic cellsex vivo, ameliorating protozoal parasitic infection, accelerate woundhealing, and protecting hair follicles.

An effective amount is defined as the amount which is required to confera therapeutic effect on the treated patient, and is typically determinedbased on age, surface area, weight, and condition of the patient. Theinterrelationship of dosages for animals and humans (based on milligramsper meter squared of body surface) is described by Freireich et al.,Cancer Chemother. Rep. 50, 219 (1966). Body surface area may beapproximately determined from height and weight of the patient. See,e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, 1970,537. An effective amount of a compound described herein can range fromabout 1 mg/kg to about 300 mg/kg. Effective doses will also vary, asrecognized by those skilled in the art, dependant on route ofadministration, excipient usage, and the possibility of co-usage,pre-treatment, or post-treatment, with other therapeutic treatmentsincluding use of other chemotherapeutic agents and radiation therapy.Other chemotherapeutic agents that can be co-administered (eithersimultaneously or sequentially) include, but not limited to, paclitaxeland its derivatives (e.g., taxotere), doxorubicin, L-asparaginase,dacarbazine, amascrine, procarbazine, hexamethylmelamine, mitoxantrone,and gemicitabine.

The pharmaceutical composition may be administered via the parenteralroute, including orally, topically, subcutaneously, intraperitoneally,intramuscularly, and intravenously. Examples of parenteral dosage formsinclude aqueous solutions of the active agent, in a isotonic saline, 5%glucose or other well-known pharmaceutically acceptable excipient.Solubilizing agents such as cyclodextrins, or other solubilizing agentswell-known to those familiar with the art, can be utilized aspharmaceutical excipients for delivery of the therapeutic compounds.Because some of the compounds described herein can have limited watersolubility, a solubilizing agent can be included in the composition toimprove the solubility of the compound. For example, the compounds canbe solubilized in polyethoxylated castor oil (Cremophor EL®) and mayfurther contain other solvents, e.g., ethanol. Furthermore, compoundsdescribed herein can also be entrapped in liposomes that may containtumor-directing agents (e.g., monoclonal antibodies having affinitytowards tumor cells).

A compound described herein can be formulated into dosage forms forother routes of administration utilizing conventional methods. Forexample, it can be formulated in a capsule, a gel seal, or a tablet fororal administration. Capsules may contain any standard pharmaceuticallyacceptable materials such as gelatin or cellulose. Tablets may beformulated in accordance with conventional procedures by compressingmixtures of a compound described herein with a solid carrier and alubricant. Examples of solid carriers include starch and sugarbentonite. Compounds of this invention can also be administered in aform of a hard shell tablet or a capsule containing a binder, e.g.,lactose or mannitol, a conventional filler, and a tableting agent.

The activities of a compound described herein can be evaluated bymethods known in the art, e.g., MTT(3-[4,5-dimehtythiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay,clonogenic assay, ATP assay, or Extreme Drug Resistance (EDR) assay. SeeFreuhauf, J. P. and Manetta, A., Chemosensitivity Testing in GynecologicMalignancies and Breast Cancer 19, 39-52 (1994). The EDR assay, inparticular, is useful for evaluating the antitumor and antiproliferativeactivity of a compound of this invention (see Example 28 below). Cellsare treated for four days with compound of the invention. Both untreatedand treated cells are pulsed with tritiated thymidine for 24 hours.Radioactivity of each type of cells is then measured and compared. Theresults are then plotted to generate drug response curves, which allowIC₅₀ values (the concentration of a compound required to inhibit 50% ofthe population of the treated cells) to be determined.

The histone acetylation activity of a compound described herein can beevaluated in an assay using mouse erythroleukemia cells. Studies areperformed with the DS19 mouse erythroleukemia cells maintained in RPMI1640 medium with 25 mM HEPES buffer and 5% fetal calf serum. The cellsare incubated at 37° C.

Histones are isolated from cells after incubation for periods of 2 and24 hours. The cells are centrifuged for 5 minutes at 2000 rpm in theSorvall SS34 rotor and washed once with phosphate buffered saline. Thepellets are suspended in 10 ml lysis buffer (10 mM Tris, 50 mM sodiumbisulfite, 1% Triton X-100, 10 mM magnesium chloride, 8.6% sucrose, pH6.5) and homogenized with six strokes of a Teflon pestle. The solutionis centrifuged and the pellet washed once with 5 ml of the lysis bufferand once with 5 ml 10 mM Tris, 13 mM EDTA, pH 7.4. The pellets areextracted with 2×1 mL 0.25N HCl. Histones are precipitated from thecombined extracts by the addition of 20 mL acetone and refrigerationovernight. The histones are pelleted by centrifuging at 5000 rpm for 20minutes in the Sorvall SS34 rotor. The pellets are washed once with 5 mLacetone and protein concentration are quantitated by the Bradfordprocedure.

Separation of acetylated histones is usually performed with an aceticacid-urea polyacrylamide gel electrophoresis procedure. Resolution ofacetylated H4 histones is achieved with 6.25N urea and no detergent asoriginally described by Panyim and Chalkley, Arch. Biochem. Biophys.130, 337-346, 1969. 25 μg total histones are applied to a slab gel whichis run at 20 ma. The run is continued for a further two hours after thePyronon Y tracking dye has run off the gel. The gel is stained withCoomassie Blue R. The most rapidly migrating protein band is theunacetylated H4 histone followed by bands with 1, 2, 3 and 4 acetylgroups which can be quantitated by densitometry. The procedure fordensitometry involves digital recording using the Alpha Imager 2000,enlargement of the image using the PHOTOSHOP program (Adobe Corp.) on aMACINTOSH computer (Apple Corp.), creation of a hard copy using a laserprinter and densitometry by reflectance using the Shimadzu CS9000Udensitometer. The percentage of H4 histone in the various acetylatedstates is expressed as a percentage of the total H4 histone.

The concentration of a compound of the invention required to decreasethe unacetylated H4 histone by 50% (i.e., EC₅₀) can then be determinedfrom data obtained using different concentrations of test compounds.

Histone deacetylase inhibitory activity can be measured based onprocedures described by Hoffmann et al., Nucleic Acids Res., 27,2057-2058 (1999). See Example 30 below. Briefly, the assay starts withincubating the isolated histone deacetylase enzyme with a compound ofthe invention, followed by the addition of a fluorescent-labeled lysinesubstrate (contains an amino group at the side chain which is availablefor acetylation). HPLC is used to monitor the labeled substrate. Therange of activity of each test compound is preliminarily determinedusing results obtained from HPLC analyses. IC₅₀ values can then bedetermined from HPLC results using different concentrations of compoundsof this invention. All assays are duplicated or triplicated foraccuracy. The histone deacetylase inhibitory activity can be comparedwith the increased activity of acetylated histone for confirmation.

Compounds of this invention are also evaluated for effects on treatingX-linked adrenoleukodystrophy (X-ALD), a peroxisomal disorder withimpaired very long-chain fatty acid (VLCFA) metabolism. In such anassay, cell lines derived from human primary fibroblasts and(EBV-transformed lymphocytes) derived from X-ALD patients grown in RPMIare employed. Tissue culture cells are grown in the presence or absenceof test compounds. For VLCFA measurements, total lipids are extracted,converted to methyl esters, purified by TLC and subjected to capillaryGC analysis as described in Moser et al., Technique in DiagnosticBiochemical Genetics: A Laboratory Manual (ed. A., H. F.) 177-191(Wiley-Liss, New York, 1991). C24:0 β-oxidation activity oflyophoclastoid cells are determined by measuring their capacity todegrade [1-¹⁴C]-C24:0 fatty acid to water-soluble products as describedin Watkins et al., Arch. Biochem. Biophys. 289, 329-336 (1991). Thestatistical significance of measured biochemical differences betweenuntreated and treated X-ALD cells can be determined by a two-tailedStudent's t-test. See Example 31 below.

Further, compounds of the present invention are evaluated for theireffects in treating cystic fibrosis (CF). Since the initial defect inthe majority of cases of CF is the inability of mutant CF protein (CFTR)to fold properly and exit the ER, compounds of the invention are testedto evaluate their efficacy in increasing the trafficking of the CFprotein out of the ER and its maturation through the Golgi. During itsbiosynthesis, CFTR is initially synthesized as a nascent polypeptidechain in the rough ER, with a molecular weight of around 120 kDa (BandA). It rapidly receives a core glycosylation in the ER, giving it amolecular weight of around 140 kDa (Band B). As CFTR exits the ER andmatures through the Golgi stacks, its glycosylation is modified until itachieves a terminal mature glycosylation, affording it a molecularweight of around 170 kDa (Band C). Thus, the extent to which CFTR exitsthe ER and traverses the Golgi to reach the plasma membrane may bereflected in the ratio of Band B to Band C protein. CFTR isimmunoprecipitated from control cells, and cells exposed to testcompounds. Both wt CFTR and ΔF508 CFTR expressing cells are tested.Following lysis, CFTR are immunoprecipitated using various CFTRantibodies. Immunoprecipitates are then subjected to in vitrophosphorylation using radioactive ATP and exogenous protein kinase A.Samples are subsequently solubilized and resolved by SDS-PAGE. Gels arethen dried and subject to autoradiography and phosphor image analysisfor quantitation of Bands B and C are determined on a BioRad personalfix image station. See Example 32 below.

Furthermore, compounds of this invention can be used to treat homozygousβ thalassemia, a disease in which there is inadequate production of βglobin leading to severe anemia. See Collins et al., Blood, 85(1), 43-49(1995).

Still further, compounds of the present invention are evaluated fortheir use as antiprotozoal or antiparasitic agents. The evaluation canbe conducted using parasite cultures (e.g., Asexual P. falciparum). SeeTrager, W. & Jensen, J. B. Science 193, 673-675 (1976). Test compoundsof the invention are dissolved in dimethyl sulfoxide (DMSO) and added towells of a flat-bottomed 96-well microtitre plate containing humanserum. Parasite cultures are then added to the wells, whereas controlwells only contain parasite cultures. After at least one invasion cycle,and addition of labeled hypoxanthine monohydrochloride, the level ofincorporation of labeled hypoxanthine is detected. IC₅₀ values can becalculated from data using a non-linear regression analysis.

The toxicity of a compound described herein is evaluated when a compoundof the invention is administered by single intraperitoneal dose to testmice. See Example 33 below. After administration of a predetermined doseto three groups of test mice and untreated controls, mortality/morbiditychecks are made daily. Body weight and gross necropsy findings are alsomonitored. For reference, see Gad, S. C. (ed.), Safety Assessment forPharmaceuticals (Van Nostrand Reinhold, New York, 1995).

Without further elaboration, it is believed that one skilled in the artcan, based on the description herein, utilize the present invention toits fullest extent. The following specific examples, which describedsyntheses, screening, and biological testing of various compounds ofthis invention, are therefore, to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. All publications recited herein, including patents, arehereby incorporated by reference in their entirety.

Example 1 Synthesis of 3-methyl-5-phenyl-2,4-pentadienoic acid

To a cooled (−10 to −5° C.) 165 mL of 3 M solution of methyl magnesiumiodide in ether was added dropwise a solution of ethyl trans-cinnamate(25.0 g) in 200 mL of anhydrous ether. The reaction was warmed to roomtemperature and stirred overnight. The mixture was then heated up to 33°C. under reflux for two hours and cooled to 0° C. A white solid wasformed during cooling and water (105 mL) was gradually added to dissolvethe white precipitate followed by an additional 245 mL of saturatedaqueous ammonium chloride solution. The mixture was then stirred untilthe solids were completely dissolved and extracted with 100 mL of etherthree times. The combined extract was washed with 100 mL of water, driedover anhydrous sodium sulfate and filtered. The solvent was evaporatedto give 22.1 g of the desired 4-phenyl-2-methyl-3-buten-2-ol as an oilwhich was used in the next step without further purification. ¹H NMR(CDCl₃, 300 MHz), δ(ppm) 7.41 (m, 5H), 6.58 (d, 1H), 6.34 (d, 1H), 1.41(broad s, 6H).

Dimethylformamide (DMF, anhydrous, 25 mL) was cooled to 0-5° C. andphosphorus oxychloride (16.4 mL) was added dropwise over a period of anhour. The resulting solution was added dropwise to a cooled (0-5° C.)solution of 4-phenyl-2-methyl-3-buten-2-ol (0.14 mol) in 60 mL ofanhydrous DMF over a period of an hour. The reaction mixture was thenwarmed to room temperature, gradually heated up to 80° C., stirred at80° C. for three hours and cooled to 0-5° C. To the cooled reactionsolution was added dropwise a solution of sodium acetate (80 g) indeionized water (190 mL) over a period of two hours. The mixture wasthen reheated to 80° C., stirred at 80° C. for an additional 10 minutes,cooled down to room temperature and extracted with ether (300 mL) twice.The combined extract was washed with water (200 mL), dried overanhydrous sodium sulfate, filtered and concentrated in vacuum to yield16.7 g of the desired 3-methyl-5-phenyl-2,4-pentadienal as a liquidwhich was used in the next step without further purification.

To a stirred solution of 3-methyl-5-phenyl-2,4-pentadienal (16.5 g) inethanol (330 mL) was added dropwise a solution of silver nitrate (19.28g) in water (160 mL) followed by dropwise addition of an aqueous sodiumhydroxide (25 g, 80 mL) solution. The resulting mixture was allowed tostir for an additional five hours and then filtered. The solid waswashed with ethanol. The combined filtrate was concentrated in vacuum.The residue was dissolved in water (200 mL). The aqueous solution wasextracted with ether (300 mL) twice and acidified with 6 N hydrochloricacid (74 mL). The solid formed was filtered and recrystallized frommethanol (40 mL) to yield 2.65 g of the desired3-methyl-5-phenyl-2,4-pentadienoic acid. ¹H NMR (acetone-d6, 300 MHz),δ(ppm) 7.60 (d, 2H), 7.35 (m, 3H), 7.06 (m, 2H), 6.02 (broad s, 1H),2.50 (s, 3H).

Example 2 Synthesis of 4-methyl-5-phenyl-2,4-pentadienoic acid

Butyllithium (135 mL of 2.5 N solution) was added to 600 mL of anhydroustetrahydrofuran (THF) at −65° C. A solution of diethylphosphonoaceticacid (30.5 g) in 220 mL of anhydrous THF was added dropwise to thestirred solution at −65° C. over a period of 60 minutes. The resultingsolution was stirred at −65° C. for an additional 30 minutes and then asolution of α-methyl-trans-cinnamaldehyde (23.2 g) in 100 mL ofanhydrous THF was added to the reaction at −65° C. over a period of 70minutes. The reaction was stirred for one hour, allowed to warm to roomtemperature and then stirred overnight. The reaction was then acidifiedwith 5% hydrochloric acid (125 mL) to a pH of 2.8. The aqueous layer wasextracted with 100 mL of ether twice and with 100 mL of ethyl acetateonce. The combined organic extract was dried over anhydrous sodiumsulfate, filtered and concentrated under vacuum. The crude material wasdissolved in 100 mL of hot methanol and then refrigerated overnight. Thecrystals formed were filtered and dried under vacuum to afford 25.8 g ofthe desired 4-methyl-5-phenyl-2,4-pentadienoic acid. ¹H NMR (acetone-d6,300 MHz), δ (ppm) 7.53 (d, 1H), 7.43 (m, 4H), 7.37 (dd, 1H), 6.97 (broads, 1H), 6.02 (d, 1H), 2.07 (s, 3H).

Example 3 Synthesis of 4-chloro-5-phenyl-2,4-pentadienoic acid

Butyllithium (50 mL of 2.5 N solution) was added to 250 mL of anhydroustetrahydrofuran (THF) at −65° C. A solution of diethylphosphonoaceticacid (11.4 g) in 90 mL of anhydrous THF was added dropwise to thestirred solution at −65° C. The resulting solution was stirred at −65°C. for an additional 40 minutes and then a solution ofα-chloro-cinnamaldehyde (10.0 g) in 60 mL of anhydrous THF was added tothe reaction at −65° C. over a period of 95 minutes. The reaction wasstirred for one hour, allowed to warm to room temperature and thenstirred overnight. The reaction was then acidified with 5% hydrochloricacid (48 mL) to a pH of 3.9. The aqueous layer was extracted with 50 mLof ether twice and with 50 mL of ethyl acetate once. The combinedorganic extract was dried over anhydrous sodium sulfate, filtered andconcentrated under vacuum. The crude material was dissolved in 30 mL ofhot methanol and then refrigerated overnight. The crystals formed werefiltered and dried under vacuum to afford 9.2 g of the desired4-chloro-5-phenyl-2,4-pentadienoic acid. ¹H NMR (acetone-d6, 300 MHz),δ(ppm) 7.86 (d, 2H), 7.60 (d, 1H), 7.45 (m, 3H), 7.36 (broad s, 1H),6.32 (d, 1H).

Example 4 Synthesis of 5-phenyl-2-ene-4-pentynoic Acid

Butyllithium (16 mL of 2.5 N solution) was added to 75 mL of anhydroustetrahydrofuran (THF) at −65° C. A solution of diethylphosphonoaceticacid (3.6 g) in 25 mL of anhydrous THF was added dropwise to the stirredsolution at −65° C. over a period of 15 minutes. The resulting solutionwas stirred at −65° C. for an additional 30 minutes and then a solutionof phenylpropargyl aldehyde (2.5 g) in 20 mL of anhydrous THF was addedto the reaction at −65° C. over a period of 20 minutes. The reaction wasstirred for one hour, allowed to warm to room temperature and thenstirred overnight. The reaction was then acidified with 6 N hydrochloricacid (5 mL) to a pH of 1.0. The aqueous layer was extracted with 75 mLof ethyl acetate three times. The combined organic extract was driedover anhydrous sodium sulfate, filtered and concentrated under vacuum.The crude material was recrystallized with chloroform:ether (90:10) andthen refrigerated overnight. The crystals were filtered and dried undervacuum to afford 1.1 g of the desired 5-phenyl-2-ene-4-pentynoic acid.¹H NMR (acetone-d6, 300 MHz), δ(ppm) 7.50 (m, 5H), 6.98 (d, 1H), 6.35(d, 1H).

Example 5 Synthesis of 5-β-dimethylaminophenyl)-2,4-pentadienoic Acid

Butyllithium (24 mL of 2.5 N solution) was added to 120 mL of anhydroustetrahydrofuran (THF) at −65° C. A solution of diethylphosphonoaceticacid (5.5 g) in 45 mL of anhydrous THF was added dropwise to the stirredsolution at −65° C. over a period of one hour. The resulting solutionwas stirred at −65° C. for an additional 30 minutes and then a solutionof p-dimethylaminocinnamaldehyde (5.0 g) in 80 mL of anhydrous THF wasadded to the reaction at −65° C. over a period of 30 minutes. Thereaction was stirred for one hour, allowed to warm to room temperatureand then stirred overnight. The reaction was then quenched with 400 mLof water and extracted with 300 mL of ethyl acetate three times. Theaqueous layer was acidified with 5% hydrochloric acid (11 mL) to a pH of6.1. The solid formed was filtered, washed with 75 mL of water and driedto yield 3.83 g of the desired5-(p-dimethylaminophenyl)-2,4-pentadienoic acid. ¹H NMR (DMSO-d₆, 300MHz), δ(ppm) 7.34 (m, 3H), 6.82 (m, 2H), 6.70 (d, 2H), 5.84 (d, 1H),2.94 (s, 6H).

Example 6 Synthesis of 5-(2-furyl)-2,4-pentadienoic acid

Butyllithium (70 mL of 2.5 N solution) was added to 350 mL of anhydroustetrahydrofuran (THF) at −65° C. A solution of diethylphosphonoaceticacid (15.9 g) in 130 mL of anhydrous THF was added dropwise to thestirred solution at −65° C. over a period of 75 minutes. The resultingsolution was stirred at −65° C. for an additional 30 minutes and then asolution of trans-3-(2-furyl)acrolein (10.0 g) in 85 mL of anhydrous THFwas added to the reaction at −65° C. over a period of 2 hours. Thereaction was allowed to warm to room temperature and stirred overnight.The reaction was then acidified with 5% hydrochloric acid (85 mL) to apH of 3.5 followed by addition of 30 mL of water. The aqueous layer wasextracted with 50 mL of ether twice and with 50 mL of ethyl acetateonce. The combined organic extract was dried over anhydrous sodiumsulfate; filtered and concentrated under vacuum to give an oil. Thecrude oil was dissolved in 45 mL of hot methanol and then refrigeratedovernight. The crystals formed were filtered and dried under vacuum toafford 9.2 g of the desired 5-(2-furyl)-2,4-pentadienoic acid. ¹H NMR(acetone-d₆, 300 MHz), δ(ppm) 7.64 (broad s, 11H), 7.42 (m, 11H), 6.86(m, 2H), 6.58 (m, 2H), 6.05 (d, 11H).

Example 7 Synthesis of 6-phenyl-3,5-hexadienoic Acid

Triphenylphosphine (178.7 g) and 3-chloropropionic acid (73.9 g) weremixed in a 1-liter 3-neck round bottom flask equipped with a mechanicalstirrer, reflux condenser with a nitrogen inlet and a thermocouple. Themixture was heated to 145° C. under nitrogen and stirred for 2 hours.The reaction was then cooled to 70° C. Ethanol (550 mL) was added andthe mixture was refluxed at 80° C. until complete dissolution. Thesolution was cooled to room temperature and ether (900 mL) was added.The mixture was placed in the freezer overnight. The solids werecollected by filtration and dried under vacuum to afford 217 g of3-(triphenylphosphonium)propionic acid chloride as a white solid whichwas used in the next step without further purification.

Sodium hydride (12.97 g) in an oven dried 5-liter 3-neck round bottomflask equipped with a mechanical stirrer and a thermocouple was cooledto 0-5° C. in an ice bath. A solution of3-(triphenylphosphonium)propionic acid chloride (100.0 g) andtrans-cinnamaldehyde (34 mL) in 400 mL each of anhydrous dimethylsulfoxide and tetrahydrofuran was added over a period of 3 hours. Thereaction was then allowed to warm to room temperature and stirredovernight. The reaction mixture was cooled to 0-5° C. in an ice bath andwater (1.6 liters) was added dropwise. The aqueous solution wasacidified with 12 N hydrochloric acid (135 mL) to a pH of 1 andextracted with ethyl acetate (1.6 liters) twice. The combined organiclayers was washed with water (1000 mL) three times, dried over anhydroussodium sulfate and concentrated under vacuum to afford a yellow oil. Thecrude oil was dissolved in 125 mL of methylene chloride andchromatographed on a Biotage 75L silica gel column and eluted withmethylene chloride:ether (9:1). The fractions containing the desiredproduct were combined and the solvents were removed under vacuum toafford 10.38 g of 6-phenyl-3,5-hexadienoic acid. ¹H NMR (CDCl₃, 300MHz), δ (ppm) 7.33 (m, 5H), 6.80 (m, 1H), 6.53 (d, 1H), 6.34 (m, 1H),5.89 (m, 1H), 3.25 (d, 2H).

Example 8 Synthesis of 7-phenyl-2,4,6-heptatrienoic Acid

To a cooled (0-55° C.) 927 mL of 1 M solution of phenyl magnesiumbromide in tetrahydofuran was added dropwise a solution ofcrotonaldehyde (65.0 g) in 130 mL of anhydrous ether over a period of 2hours and 45 minutes. The reaction was stirred for an additional 45minutes and then warmed to room temperature. After four more hours ofstirring, saturated ammonium chloride aqueous solution (750 mL) wasadded to the reaction. The mixture was extracted with 750 mL of ethertwice. The combined extract was dried over anhydrous potassium carbonateand filtered. The solvent was evaporated to give 135.88 g (99.9%) of thedesired 1-phenyl-2-buten-1-ol as an oil which was used in the next stepwithout further purification.

1-Phenyl-2-buten-1-ol (135.88 g) was dissolved in 2300 mL of dioxane andtreated with 2750 mL of dilute hydrochloric acid (2.3 mL of concentratedhydrochloric acid in 2750 mL of water) at room temperature. The mixturewas stirred overnight and then poured into 4333 μL of ether andneutralized with 2265 mL of saturated aqueous sodium bicarbonate. Theaqueous phase was extracted with 1970 mL of ether. The combined extractwas dried over anhydrous potassium carbonate. Evaporation of the solventfollowed by Kugelrohr distillation at 30° C. for 30 minutes afforded131.73 g (96.8%) of the desired 4-phenyl-3-buten-2-ol as an oil whichwas used in the next step without further purification.

Dimethylformamide (DMF, anhydrous, 14 mL) was cooled to 0-5° C. andphosphorus oxychloride (8.2 mL) was added dropwise over a period of 40minutes. The resulting solution was added dropwise to a cooled (0-5° C.)solution of 4-phenyl-3-buten-2-ol (10 g) in 32 mL of anhydrous DMF overa period of an hour. The reaction mixture was warmed to room temperatureover a 35-minute period and then gradually heated up to 80° C. over aperiod of 45 minutes. The reaction was stirred at 80° C. for three hoursand then cooled to 0-5° C. To the cooled reaction solution was addeddropwise a solution of sodium acetate (40 g) in deionized water (100 mL)over a period of one hour. The mixture was then reheated to 80° C.,stirred at 80° C. for an additional 10 minutes, cooled down to roomtemperature and extracted with ether (100 mL) twice. The combinedextract was washed with brine (100 mL), dried over anhydrous sodiumsulfate, filtered and concentrated under vacuum to yield 8.78 g of thedesired 5-phenyl-2,4-pentadienal as a liquid which was used in the nextstep without further purification. ¹H NMR (CDCl₃, 300 MHz), δ(ppm) 7.51(m, 2H), 7.37 (m, 3H), 7.26 (m, 1H), 7.01 (m, 2H), 6.26 (m, 1H).

Butyllithium (12.8 mL of 2.5 N solution) was added to 65 mL of anhydroustetrahydrofuran (THF) at −65° C. A solution of diethylphosphonoaceticacid (2.92 g) in 25 mL of anhydrous THF was added dropwise to thestirred solution at −65° C. The resulting solution was stirred at −65°C. for an additional 30 minutes and then a solution of5-phenyl-2,4-pentadienal (2.4 g) in 15 mL of anhydrous THF was added tothe reaction at −65° C. The reaction was stirred for one hour, allowedto warm to room temperature and then stirred overnight. To the reactionwas added 30 mL of water, acidified with 5% hydrochloric acid (14 mL) toa pH of 4.7 and then added an additional 20 mL of water. The aqueouslayer was extracted with 10 mL of ether twice and with 10 mL of ethylacetate once. The combined organic extract was dried over anhydroussodium sulfate, filtered and concentrated under vacuum. The crudematerial was dissolved in 50 mL of hot methanol and then refrigeratedovernight. The crystals formed were filtered and dried under vacuum toafford 2.4 g of the desired 7-phenyl-2,4,6-heptatrienoic acid. ¹H NMR(DMSO-d₆, 300 MHz), δ(ppm) 7.52 (m, 2H), 7.33 (m, 4H), 7.06 (m, 1H),6.86 (m, 2H), 6.58 (m, 1H), 5.95 (d, 1H).

Example 9 Synthesis of 8-phenyl-3,5,7-octatrienoic Acid

A solution of 5-phenyl-2,4-pentadienal (15 g) and3-(triphenylphosphonium)-propionic acid chloride (35.2 g) in 140 mL eachof anhydrous tetrahydrofuran and anhydrous dimethyl sulfoxide was addeddropwise to sodium hydride (4.6 g) at 0-5° C. under nitrogen over aperiod of four hours. The reaction was allowed to warm to roomtemperature and stirred overnight. The reaction mixture was cooled to0-5° C. and water (280 mL) was added dropwise over a period of 30minutes. The aqueous layer was extracted with ethyl acetate (280 mL)twice, acidified with 12 N hydrochloric acid (24 mL) to a pH of 1,extracted again with ethyl acetate (280 mL) twice. The combined organiclayers were washed with water (500 mL) twice, dried over anhydroussodium sulfate and concentrated under vacuum to give an oil. The oilycrude product was chromatographed on a Biotage 40M silica gel column andeluted with methylene chloride:ethyl acetate (95:5). The fractionscontaining the desired product were combined and the solvents wereremoved under vacuum to afford 0.7 g of 8-phenyl-3,5,7-octatrienoicacid. ¹H NMR (acetone-d6, 300 MHz), δ(ppm) 7.46 (m, 2H), 7.26 (m, 3H),6.95 (m, 1H), 6.60 (d, 1H), 6.34 (m, 3H), 5.87 (m, 1H), 3.17 (d, 2H).

Example 10 Synthesis of potassium 2-oxo-6-phenyl-3,5-hexadienoate

A solution of trans-cinnamaldehyde (26.43 g) and pyruvic acid (11.9 mL)in 10 mL of methanol was stirred and chilled to 0-5° C. in an ice bath.To the chilled solution was added 35 nL of potassium hydroxide (16.83 gin 50 mL of methanol) over a period of 20 minutes. The remainingmethanolic potassium hydroxide was added rapidly and the ice bath wasremoved. The solution changed from a yellow to a dark orange and theprecipitate was formed. The reaction mixture was chilled in therefrigerator overnight and the solid was collected by filtration, washedwith 50 mL of methanol three times, 50 mL of ether and then air dried toafford 29.3 g of the desired 2-oxo-6-phenyl-3,5-hexadienoate as a yellowsolid (61.0%). ¹H NMR (DMSO-d₆/D₂O, 300 MHz), δ(ppm) 7.48 (d, 2H), 7.28(m, 4H), 7.12 (d, 2H), 6.27 (d, 1H).

Example 11 Synthesis of Potassium 2-oxo-8-phenyl-3,5,7-octatrienoate

To a cooled (0-55° C.) 927 mL of 1 M solution of phenyl magnesiumbromide in tetrahydrofuran was added dropwise a solution ofcrotonaldehyde (65.0 g) in 130 μL of anhydrous ether over a period of 2hours and 45 minutes. The reaction was stirred for an additional 45minutes and then warmed to room temperature. After four more hours ofstirring, saturated ammonium chloride aqueous solution (750 mL) wasadded to the reaction. The mixture was extracted with 750 mL of ethertwice. The combined extract was dried over anhydrous potassium carbonateand filtered. The solvent was evaporated to give 135.88 g (99.9%) of thedesired 1-phenyl-2-buten-1-ol as an oil which was used in the next stepwithout further purification.

1-Phenyl-2-buten-1-ol (135.88 g) was dissolved in 2300 mL of dioxane andtreated with 2750 mL of dilute hydrochloric acid (2.3 mL of concentratedhydrochloric acid in 2750 mL of water) at room temperature. The mixturewas stirred overnight and then poured into 4333 mL of ether andneutralized with 2265 mL of saturated sodium bicarbonate. The aqueousphase was extracted with 1970 mL of ether. The combined extract wasdried over anhydrous potassium carbonate. Evaporation of the solventfollowed by Kugelrohr distillation at 30° C. for 30 minutes afforded131.73 g (96.8%) of the desired 4-phenyl-3-buten-2-ol as an oil whichwas used in the next step without further purification.

Dimethylformamide (DMF, anhydrous, 14 mL) was cooled to 0-5° C. andphosphorus oxychloride (8.2 m/L) was added dropwise over a period of 40minutes. The resulting solution was added dropwise to a cooled (0-5° C.)solution of 4-phenyl-3-buten-2-ol (10 g) in 32 mL of anhydrous DMF overa period of an hour. The reaction mixture was warmed to room temperatureover a 35-minute period and then gradually heated up to 80° C. over aperiod of 45 minutes. The reaction was stirred at 80° C. for three hoursand then cooled to 0-5° C. To the cooled reaction solution was addeddropwise a solution of sodium acetate (40 g) in deionized water (100 mL)over a period of one hour. The mixture was then reheated to 80° C.,stirred at 80° C. for an additional 10 minutes, cooled down to roomtemperature and extracted with ether (100 mL) twice. The combinedextract was washed with brine (100 mL), dried over anhydrous sodiumsulfate, filtered and concentrated under vacuum to yield 8.78 g of thedesired 5-phenyl-2,4-pentadienal as a liquid which was used in the nextstep without further purification. ¹H NMR (CDCl₃, 300 MHz), δ(ppm) 7.51(m, 2H), 7.37 (m, 3H), 7.26 (m, 1H), 7.01 (m, 2H), 6.26 (m, 1H).

A solution of 5-phenyl-2,4-pentadienal (6.70 g) and pyruvic acid (3.0mL) in 5 mL of methanol was stirred and chilled to 0-5° C. in an icebath. To the chilled solution was added a solution of 35 mL of potassiumhydroxide (3.5 g) in 10 mL of methanol dropwise over a period of 30minutes. The remaining methanolic potassium hydroxide was added rapidlyand the ice bath was removed. The reaction was allowed to warm to roomtemperature and stirred for another hour. The flask was thenrefrigerated overnight. The solid was collected by filtration, washedwith 15 mL of methanol three times, 15 mL of ether and then air dried toafford 6.69 g of potassium 2-oxo-8-phenyl-3,5,7-octatrienoate as ayellow solid. ¹H NMR (DMSO-d, 300 MHz), δ(ppm) 7.52 (d, 2H), 7.32 (m,3H), 7.10 (m, 2H), 6.83 (dd, 2H), 6.57 (dd, 1H), 6.13 (d, 1H).

Example 12 Synthesis of Cinnamoylhydroxamic Acid

Triethylamine (TEA, 17.6 mL) was added to a cooled (0-5° C.) solution oftrans-cinnamic acid (15.0 g) in 200 mL of anhydrous dimethylformamide.To this solution was added dropwise isobutyl chloroformate (16.4 mL).The reaction mixture was stirred for 30 minutes and hydroxylaminehydrochloride (17.6 g) was added followed by dropwise addition of 35 mLof TEA at 0-5° C. The reaction was allowed to warm to room temperatureand stirred overnight. The reaction was quenched with 250 mL of 1% (byweight) citric acid solution and 50 mL of 5% (by weight) citric acidsolution and then extracted with 200 mL of methylene chloride twice and200 mL of ether once. The solvents were removed under vacuum. Theresidue was triturated with 125 mL of water, filtered, washed with 25 mLof water and dried under vacuum to give a tan solid. The crude productwas chromatographed on a Biotage 75S column and eluted with methylenechloride:acetonitrile (80:20). The fractions containing the desiredproduct were combined and the solvent was removed under vacuum to yield4.1 g of cinnamoylhydroxamic acid. ¹H NMR (DMSO-d₆, 300 MHz), δ(ppm)7.48 (m, 6H), 6.49 (d, 1H).

Example 13 Synthesis of N-methyl-Cinnamoylhydroxamic Acid

A solution of cinnamoyl chloride (5 g) in 50 mL of methylene chloridewas added dropwise to a solution of N-methylhydroxylamine hydrochloride(5 g) and 12 mL of 40% sodium hydroxide in 50 mL of water cooled to 0-5°C. The reaction mixture was stirred for two hours. The aqueous layer wasacidified with concentrated hydrochloric acid. The precipitate wascollected by filtration and dried under vacuum to afford 2.8 g of thedesired N-methyl-cinnamoylhydroxamic acid as a white solid. ¹H NMR(DMSO-d₆, 300 MHz), δ (ppm) 7.66 (d, 2H), 7.53 (d, 1H), 7.42 (m, 3H),7.26 (d, 1H), 3.22 (s, 3H).

Example 14 Synthesis of 5-phenyl-2,4-pentadienoylhydroxamic Acid

Triethylamine (TEA, 29 mL) was added to a cooled (0-5° C.) solution of5-phenyl-2,4-pentadienoic acid (29.0 g) in 300 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (27.0 mL). The reaction mixture was stirred for 15 minutesand hydroxylamine hydrochloride (28.92 g) was added followed by dropwiseaddition of 58 mL of TEA over a period of 60 minutes at 0-5° C. Thereaction was allowed to warm to room temperature and stirred overnight.The reaction was then poured into 450 mL of a 1% (by weight) solution ofcitric acid and then extracted with 200 mL of methylene chloride twiceand 500 mL of ether once. The solvents were removed under vacuum to givean oil. The crude oil was crystallized with 200 mL of hot acetonitrileto give a tan solid. The tan solid was recrystallized from 60 mL of hotacetonitrile to afford 12.5 g of the desired5-phenyl-2,4-pentadienoylhydroxamic acid. ¹H NMR (DMSO-d₆, 300 MHz), δ(ppm) 7.56 (d, 2H), 7.31 (m, 4H), 7.03 (m, 2H), 6.05 (s, 1H).

Example 15 Synthesis of N-methyl-5-phenyl-2,4-pentadienoylhydroxamicAcid

5-Phenyl-2,4-pentadienoic acid (6 g) and oxalyl chloride (6.1 mL) weredissolved in 50 mL of methylene chloride and 0.2 mL of dimethylformamidewas added. The reaction was stirred for three hours, concentrated undervacuum and then co-evaporated with 100 mL of chloroform to remove oxalylchloride. The crude 5-phenyl-2,4-pentadienoic acid chloride was used inthe next step without further purification.

5-Phenyl-2,4-pentadienoic acid chloride was dissolved in 50 mL ofmethylene chloride and added to a solution of 13.8 mL of 40% sodiumhydroxide in 50 mL of water at 0-5° C. The resulting solution wasstirred for two hours and then acidified to a pH of 4 with concentratedhydrochloric acid. The precipitate was collected by filtration and driedunder vacuum to afford 4.2 g ofN-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid. ¹H NMR (DMSO-d, 300MHz), δ(ppm) 7.57 (d, 2H), 7.35 (m, 4H), 7.19 (m, 1H), 6.99 (d, 1H),6.82 (d, 1H), 3.21 (s, 3H).

Example 16 Synthesis of 3-methyl-5-phenyl-2,4-pentadienoylhydroxamicacid

Triethylamine (TEA, 1.8 mL) was added to a cooled (0-5° C.) solution of3-methyl-5-phenyl-2,4-pentadienoic acid (2.0 g) in 20 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (1.7 mL) over a period of 15 minutes. The reaction mixturewas stirred for 30 minutes and hydroxylamine hydrochloride (1.85 g) wasadded followed by dropwise addition of 3.7 mL of TEA over a period of 35minutes at 0-5° C. The reaction was allowed to warm to room temperatureand stirred overnight. To the stirred reaction mixture at roomtemperature was added 20 mL of a 1% (by weight) solution of citric acidfollowed by 75 mL of water. The mixture was stirred for 30 minutes andthen filtered. The filtered cake was washed with 30 mL of water anddried in vacuum to afford 1.49 g of the desired3-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid in 69% yield. ¹H NMR(DMSO-d, 300 MHz), δ(ppm) 7.55 (d, 2H), 7.30 (m, 3H), 6.89 (broad s,2H), 5.83 (s, 1H), 2.38 (s, 3H).

Example 17 Synthesis of 4-methyl-5-phenyl-2,4-pentadienoylhydroxamicAcid

Triethylamine (TEA, 6.5 mL) was added to a cooled (0-5° C.) solution of4-methyl-5-phenyl-2,4-pentadienoic acid (7.0 g) in 75 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (6.0 mL) over a period of 60 minutes. The reaction mixturewas stirred for 15 minutes and hydroxylamine hydrochloride (6.5 g) wasadded followed by dropwise addition of 13 mL of TEA over a period of 60minutes at 0-5° C. The reaction was allowed to warm to room temperatureand stirred overnight. To the stirred reaction mixture at roomtemperature was added 130 mL of a 1% (by weight) solution of citric acidfollowed by 50 mL of water. The mixture was stirred for 30 minutes andthen filtered. The filtered cake was recrystallized from hotacetonitrile to afford 4.4 g of the desired4-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid. ¹H NMR (DMSO-d₆, 300MHz), δ(ppm) 7.37 (m, 6H), 6.91 (s, 1H), 6.02 (d, 1H), 1.99 (s, 3H).

Example 18 Synthesis of 4-chloro-5-phenyl-2,4-pentadienoylhydroxamicacid

Triethylamine (TEA, 2.5 mL) was added to a cooled (0-5° C.) solution of4-chloro-5-phenyl-2,4-pentadienoic acid (3.0 g) in 30 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (2.3 mL) over a period of 15 minutes. The reaction mixturewas stirred for 30 minutes and hydroxylamine hydrochloride (2.5 g) wasadded followed by dropwise addition of 5.0 mL of TEA over a period of 60minutes at 0-5° C. The reaction was allowed to warm to room temperatureand stirred overnight. The reaction was then quenched with 30 mL of a 1%(by weight) solution of citric acid followed by 115 mL of water. Themixture was stirred for 30 minutes and then filtered. The filtered cakewas washed with 100 mL of water and dried under vacuum. The crudematerial was recrystallized from 20 mL of hot acetonitrile twice toyield 1.46 g of the desired 4-chloro-5-phenyl-2,4-pentadienoylhydroxamicacid as a solid. ¹H NMR (DMSO-d₆, 300 MHz), δ(ppm) 7.75 (d, 2H), 7.40(m, 5H), 6.31 (d, 1H).

Example 19 Synthesis of 5-phenyl-2-ene-4-pentynoylhydroxamic acid

Triethylamine (TEA, 1.1 mL) was added to a cooled (0-5° C.) solution of5-phenyl-2-ene-4-pentynoic acid (1.1 g) in 13 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (1.0 mL). The reaction mixture was stirred for 30 minutesand hydroxylamine hydrochloride (1.1 g) was added followed by dropwiseaddition of 2.2 mL of TEA at 0-5° C. The reaction was allowed to warm toroom temperature and stirred overnight. The reaction was quenched with15 mL of a 1% (by weight) solution of citric acid and extracted with 30mL of methylene chloride twice. The combined organic layer was driedover anhydrous sodium sulfate. The solvents were removed under vacuum togive an oil which in turn was triturated with 10 mL of chloroform. Thesolid was collected by filtration to yield 0.63 g of the desired5-phenyl-2-ene-4-pentynoylhydroxamic acid as a white powder. ¹H NMR(DMSO-d₆, 300 MHz), δ(ppm) 7.48 (m, 5H), 6.76 (d, 1H), 6.35 (d, 1H).

Example 20 Synthesis of5-(p-dimethylaminophenyl)-2,4-pentadienoylhydroxamic acid

Triethylamine (TEA, 0.8 mL) was added to a cooled (0-5° C.) solution of5-(p-dimethylaminophenyl)-2,4-pentadienoic acid (1.0 g) in 10 mL ofanhydrous dimethylformamide. To this solution was added dropwiseisobutyl chloroformate (0.7 mL). The reaction mixture was stirred for 60minutes and hydroxylamine hydrochloride (0.8 g) was added followed bydropwise addition of 1.6 mL of TEA at 0-5° C. The reaction was allowedto warm to room temperature and stirred overnight. The reaction wasquenched with 15 mL of water. The solid was filtered and dried undervacuum to yield 0.75 g of the desired5-(p-dimethylaminophenyl)-2,4-pentadienoylhydroxamic acid. ¹H NMR(DMSO-d₆, 300 MHz), δ(ppm) 7.33 (m, 3H), 6.86 (m, 2H), 6.70 (d, 2H),5.84 (d, 1H), 2.99 (s, 6H).

Example 21 Synthesis of 5-(2-furyl)-2,4-pentadienoylhydroxamic acid

Triethylamine (TEA, 2.1 mL) was added to a cooled (0-5° C.) solution of5-(2-furyl)-2,4-pentadienoic acid (2.0 g) in 15 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (2.0 mL) over a period of 30 minutes. The reaction mixturewas stirred for 30 minutes and hydroxylamine hydrochloride (2.15 g) wasadded followed by dropwise addition of 4.2 mL of TEA over a period of 60minutes at 0-5° C. The reaction was allowed to warm to room temperatureand stirred overnight. To the stirred reaction mixture at roomtemperature was added 12 mL of a 1% (by weight) solution of citric acidfollowed by 46 mL of water. The mixture was stirred for 30 minutes andthen filtered. The filtered cake was washed with 30 mL of water anddried in vacuum to afford 1.3 g of the desired5-(2-furyl)-2,4-pentadienoylhydroxamic acid. ¹H NMR (DMSO-d₆, 300 MHz),δ (ppm) 7.73 (broad s, 1H), 7.22 (m, 1H), 6.71 (m, 4H), 6.01 (d, 1H).

Example 22 Synthesis of 6-phenyl-3,5-hexadienoylhydroxamic acid

Triethylamine (TEA, 1.75 mL) was added to a cooled (0-5° C.) solution of6-phenyl-3,5-hexadienoic acid (2.0 g) in 30 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (1.62 mL) over a period of 15 minutes. The reactionmixture was stirred for 15 minutes and hydroxylamine hydrochloride (1.74g) was added followed by dropwise addition of 3.5 mL of TEA at 0-5° C.The reaction was allowed to warm to room temperature and stirredovernight. The reaction was then poured into 20 mL of 1% (by weight)aqueous citric acid solution and extracted with 20 mL of methylenechloride twice and ether once. The combined organic layer was dried overanhydrous sodium sulfate and concentrated under vacuum to give a darkred oil. The crude oil was crystallized with 10 mL of hot acetonitrile.The solid was collected by filtration and then purified on a Biotage 40Ssilica gel column using methylene chloride:ether (95:5) as an eluent.The fractions containing the desired product were combined and thesolvent was removed to give 40 mg of 6-phenyl-3,5-hexadienoylhydroxamicacid as a tan solid (2.1%). ¹H NMR (DMSO-d₆, 300 MHz), δ(ppm) 7.34 (m,5H), 6.91 (m, 1H), 6.55 (d, 1H), 6.30 (m, 1H), 5.89 (m, 1H), 3.36 (d,2H).

Example 23 Synthesis of N-methyl-6-phenyl-3,5-hexadienoylhydroxamic acid

6-Phenyl-3,5-hexadienoic acid (1 g) was dissolved in 10 mL oftetrahydrofuran (THF) and treated with 0.9 g of1,1′-carbonyldiimidazole. The reaction was stirred for 30 minutes.N-methylhydroxylamine hydrochloride (0.44 g) was neutralized with 0.29 gof sodium methoxide in 10 mL of THF and 5 mL of methanol and thenfiltered to remove the sodium chloride. N-methylhydroxylamine was thenadded to the reaction mixture and stirred overnight. The resultingmixture was partitioned between 25 mL of water and 50 mL of ethylacetate. The ethyl acetate layer was washed with 25 mL each of 5%hydrochloric acid, saturated sodium bicarbonate and brine, dried oversodium sulfate and concentrated under vacuum to afford 0.9 g of aviscous yellow oil. The crude product was chromatographed on a Biotage40S silica gel column and eluted with ethyl acetate:hexane (1:1). Thefractions containing the desired product were combined and the solventwas removed under vacuum to yield 0.17 g ofN-methyl-6-phenyl-3,5-hexadienoylhydroxamic acid. ¹H NMR (CDCl₃, 300MHz), δ(ppm) 7.38 (m, 5H), 6.80 (m, 1H), 6.60 (m, 1H), 6.35 (m, 1H),5.89 (m, 1H), 3.24 (m, 2H), 2.92 (s, 3H).

Example 24 Synthesis of 7-phenyl-2,4,6-heptatrienoylhydroxamic acid

Triethylamine (TEA, 24.1 mL) was added to a cooled (0-5° C.) solution of7-phenyl-2,4,6-heptatrienoic acid (27.8 g) in 280 mL of anhydrousdimethylformamide. To this solution was added dropwise isobutylchloroformate (22.5 mL) over a period of 75 minutes. The reactionmixture was stirred for 40 minutes and hydroxylamine hydrochloride (24.2g) was added followed by dropwise addition of 48 mL of TEA over a periodof 70 minutes at 0-5° C. The reaction was allowed to warm to roomtemperature and stirred overnight. To the stirred reaction mixture atroom temperature was added 280 mL of a 1% (by weight) solution of citricacid followed by 1050 mL of water. The mixture was stirred for 30minutes and then filtered. The filtered cake was washed with water (200mL) and dried under vacuum to afford 20.5 g of the desired7-phenyl-2,4,6-heptatrienoylhydroxamic acid. ¹H NMR (DMSO-d6, 300 MHz),δ(ppm) 7.48 (m, 2H), 7.32 (m, 2H), 7.19 (m, 2H), 7.01 (m, 1H), 6.75 (m,2H), 6.51 (m, 1H), 5.93 (d, 1H).

Example 25 Synthesis of 4-cyclohexylbutyroylhydroxamic acid

To a solution of hydroxylamine hydrochloride (7.3 g) in 50 mL ofmethanol was added 24 mL of sodium methoxide (25% wt.) dropwise at roomtemperature over a period of 45 minutes. To this solution was addedmethyl 4-cyclohexylbutyrate in 50 mL of methanol at room temperaturefollowed by 12 mL of sodium methoxide (25% wt.) dropwise over a periodof 60 minutes. The resulting mixture was stirred at room temperatureovernight. The reaction was then poured into 120 mL of water andacidified to a pH of 4 with 45 mL of glacial acetic acid. Methanol wasremoved under vacuum. The solid formed was filtered and dried overphosphorus pentoxide to afford 8.53 g of the desired4-cyclohexylbutyroyl-hydroxamic acid. ¹H NMR (DMSO-d₆, 300 MHz), δ(ppm)3.38 (m, 2H), 1.91 (t, 2H), 1.68 (m, 4H), 1.50 (m, 2H), 1.16 (m, 5H),0.84 (m, 2H).

Example 26 Synthesis of S-benzylthioglycoloylhydroxamic Acid

S-benzylthioglycolic acid (12.0 g) was dissolved in 250 mL of methanoland sparged with hydrogen chloride gas at room temperature for 20minutes. The solvent was then removed under vacuum. MethylS-benzylthioglycolate obtained was used in the next step without furtherpurification.

To a solution of hydroxylamine hydrochloride (9.2 g) in 60 mL ofmethanol was added 30 mL of sodium methoxide (25% wt.) dropwise at roomtemperature over a period of 30 minutes. To this solution was addedmethyl S-benzylthioglycolate in 50 mL of methanol at room temperaturefollowed by 15 mL of sodium methoxide (25% wt.) dropwise over a periodof 60 minutes. The resulting mixture was stirred at room temperatureovernight. The reaction was then poured into 150 mL of water andacidified to a pH of 4 with 55 mL of glacial acetic acid. Methanol wasremoved under vacuum. The solid formed was filtered and dried overphosphorus pentoxide to afford 8.57 g of the desiredS-benzylthioglycoloyl-hydroxamic acid. ¹H NMR (DMSO-d₆, 300 MHz), δ(ppm)7.29 (m, 5H), 3.84 (s, 2H), 2.93 (s, 2H).

Example 27 Synthesis of 5-phenylpentanoloylhydroxamic Acid

5-Phenylpentanoic acid (10.0 g) was dissolved in 250 mL of methanol andsparged with hydrogen chloride gas at room temperature for 15 minutes.The solvent was then removed under vacuum. Methyl 5-phenylpentanoateobtained was used in the next step without further purification.

To a solution of hydroxylamine hydrochloride (7.8 g) in 50 mL ofmethanol was added 26 mL of sodium methoxide (25% wt.) dropwise at roomtemperature over a period of 45 minutes. To this solution was addedmethyl 5-phenylpentanoate in 50 mL of methanol at room temperaturefollowed by 15 mL of sodium methoxide (25% wt.) dropwise over a periodof 60 minutes. The resulting mixture was stirred at room temperatureovernight. The reaction was then poured into 150 mL of water andacidified to a pH of 4 with 40 mL of glacial acetic acid. The solventswere removed under vacuum to give a yellow oil. The yellow oil wasplaced on a Biotage 40M silica gel column and eluted with methylenechloride:ethanol (95:5). The fractions containing the desired product asindicated by the NMR were combined. The solvents were removed undervacuum to afford 8.30 g of the desired 5-phenylpentanoylhydroxamic acid.¹H NMR (DMSO-d₆, 300 MHz), δ(ppm) 7.22 (m, 5H), 3.42 (s, 3H), 2.55 (t,2H), 1.98 (t, 2H), 1.52 (m, 4H).

Example 28 In vitro Efficacy Studies Extreme Drug Resistance (EDR) Assay

The PC3 cell line was maintained in RPM1 supplemented with 10% fetalcalf serum and antibiotics. Cells were suspended in 0.12% soft agar incomplete medium and plated (2,000 cells per well) in different drugconcentrations onto a 0.4% agarose underlayer in 24-well plates. Platingcalls on agarose underlayers supports the proliferation only of thetransformed cells, ensuring that the growth signal stems from themalignant component of the tumor.

All compounds were dissolved were dissolved in DMSO to 200× stocksolutions. Stock solutions were diluted to 20× working solutions usingthe tissue culture medium, serially diluted and added to the 24-wellplates. The initial range of concentrations was 1 micromolar to 200micromolar. This concentration range was extended in the case ofN-methyl-5-phenyl-2,4-pentadienoylhydroxamic acid to 10 μM-500 μM and inthe case of tricostatin A to 0.001 μM to 0.3 μM. No significant changesin pH of the culture medium were observed under the above conditions.Diluent control wells contained PC3 cells treated with DMSO, at thedilutions used for appropriate drug treatment. All experimental pointswere represented by two separate wells (duplicates). Four wellscontaining tumor cells that were not treated with drugs served asnegative controls in each experiment.

Cells were incubated with drugs under standard culture conditions for 5days. Cultures were pulsed with tritiated thymidine (3H-TdR, New LifeScience Products, Boston, Mass.) at 5 μCi per well for the last 48 hoursof the culture period. Cell culture plates were then heated to 90° C. toliquefy the agarose, and cells were harvested onto glass fiber filters,which were then placed into counting vials containing liquidscintillation fluid. The radioactivity trapped on the filters wascounted with a Beckman scintillation counter. The fraction of survivingcells was determined by comparing ³H-TdR incorporation in treated(experimental points) and untreated (negative control) wells. MicrosoftExcel was used to organize the raw data on EDR experiments, and theSigmaPlot program was utilized to generate drug response curves. Alldrug response curves were as approximated as sigmoidal equations(characteristic for typical drug response curves) to fit the data. IC₅₀values were determined using the approximated sigmoidal curves andexpressed as mM.

IC₅₀ values of the test compounds of the invention range fromapproximately 1 μM to approximately 2000 μM.

Example 29 Histone (Hyper)Acetylation Assay

The model used in this assay was mouse erythroleukemia cells.Specifically, the level of acetylation of H4 histones in theseerythroleukemia cells was monitored. H4 histones was chosen as thetarget due to the ease of resolution of the variably acetylatedhistones. Inhibition of histone deacetylase leads to increased(hyper)acetylation of histones. Activities on histone deacetylase wereexamined to confirm the results of this assay. See Example 30 below.

Studies were performed with the DS19 mouse erythroleukemia cellsmaintained in RPMI 1640 medium with 25 mM HEPES buffer and 5% fetal calfserum. The cells were incubated at 37° C. In studies on proliferation,cell density was determined at 24 hour intervals using a hemacytometer.

Histone Isolation

Histones were isolated from cells after incubation for 2 or 24 hours.The cells were centrifuged for 5 minutes at 2,000 rpm in the SorvallSS34 rotor and washed once with phosphate buffered saline. The pelletswere suspended in 5 mL lysis buffer (10 mM Tris, 50 mM sodium bisulfite,1% Triton X-100, 10 mM magnesium chloride, 8.6% sucrose, pH 6.5) andhomogenized with six strokes of a teflon pestle. The homogenizing tubeswere rinsed with 5 mL lysis buffer. The combined solutions werecentrifuged and the pellets were washed once with 5 mL of the lysisbuffer and once with 5 mL 10 mM Tris, 13 mM EDTA, pH 7.4. The pelletswere extracted with 2×1 mL 0.25N HCl. Histones were precipitated fromthe combined extracts by the addition of 20 mL acetone and refrigerationovernight. The histones were pelleted by centrifuging at 5,000 rpm for20 minutes in the Sorvall SS34 rotor. The pellets were washed once with5 mL acetone and protein concentration was quantitated by the Bradfordprocedure.

Polyacrylamide Gel Electrophoresis

Separation of acetylated histones was performed with an acetic acid-ureapolyacrylamide gel electrophoresis procedure as originally described byPanyim and Chalkley Arch. Biochem. Biophys. 130, 337-346, 1969. 25 μghistones were applied to a slab gel which was run at 20 ma. The run wascontinued for a further two hours after the Pyronin Y tracking dye hadrun off the gel. The gel was stained with Coomassie Blue R. The mostrapidly migrating protein band is the unacetylated H4 histone followedby bands with 1, 2, 3 and 4 acetyl groups which were quantitated bydensitometry.

Densitometry

Densitometry was measured through digital recording using the AlphaImager 2000. Enlargement of the image was done using PHOTOSHOP (AdobeCorp.) on a MACINTOSH (Apple Corp.) computer. After creating a hard copyof the gel by using a laser printer, a Shimadzu CS9OOOU densitometer wasused to measure densitometry by reflectance. The percentage of H4histone in the various acetylated states was expressed as a percentageof the total H4 histone.

Results

Many of the test compounds of the invention showed EC₅₀ values inmicromolar concentration range.

Example 30 Histone Deacetylation Assay

The determination of the inhibition of histone deacetylase by compoundsof the invention was based upon the procedure described by Hoffmann etal., Nucleic Acids Res. 27, 2057-2058 (1999). The histone deacetylasewas isolated from rat liver as previously described in Kolle, D. et al.,Methods: A Companion to Methods in Enzymology 15, 323-331 (1998).Compounds were initially dissolved in either ethanol or in DMSO toprovide a working stock solution. The synthetic substrate used in theassay isN-(4-methyl-7-coumarinyl)-N-α(tert-butyloxy-carbonyl)-N-Ω-acetyllysineamide(MAL).

The assay was performed in a final total volume of 120 μL consisting of100 μL of 15 mM tris-HCl buffer at pH 7.9 and 0.25 mM EDTA, 10 mM NaCl,10% glycerol, 10 mM mercaptoethanol and the enzyme. The assay wasinitiated upon the addition of 10 μl of a test compound followed by theaddition of a fluorescent-labeled lysine substrate to each assay tube inan ice bath for 15 minutes. The tubes were transferred to a water bathat 37° C. for an additional 90 minutes.

An initial assay was performed to determine the range of activity ofeach test compound. The determination of IC₅₀ values was made from theresults of five dilutions in range according to the expected potency foreach test compound. Each assay was duplicated or triplicated.

Test compounds of the invention showed potent inhibition of histonedeacetylase, having IC₅₀ values in the low micromolar concentrationrange (e.g., two test compounds showed IC₅₀ values of 1.7 μM and 1.8μM).

Example 31 X-ALD Screening Assay

Cell Cultures and Drug Treatment

Cell lines derived from X-ALD human patients were grown in RPMIsupplemented with fetal calf serum (10%), penicillin (100 U/mL),streptomycin (100 U/mL) and glutamine (2 mM). On day 0, cells weredivided into two separate tissue culture flasks, and test compounds(2.5-250 μM final concentration, diluted from a 0.5 M stock solution inPBS, pH 7.6) was added to one flask. Cells in the second flask weregrown in the absence of test compounds for the same length of time andserved as controls. The media were changed every 3-4 days.

Biochemical Measurements

As described above, tissue culture cells were grown in the presence orabsence of test compounds, collected from tissue culture flasks usingtrypsin, washed twice with PBS and subjected to biochemical analysis.VLCFA measurements was conducted by extracting total amount of lipids,converted the lipids to methyl ester, purified by TLC, and subjected tocapillary CC analysis as described in Moser et al., Technique inDiagnostic Biochemical Genetics: A Laboratory Manual (ed. A., H. F.)177-191 (Wiley-Liss, New York, 1991). Duplicate assays were set upindependently and were assayed on different days. C24:0-oxidationactivity of lymphoblastoid cells was determined by measuring theircapacity to degrade [1-¹⁴C]-C24:0 fatty acid to water-soluble productsas described in Watkins et al., Arch. Biochem. Biophys. 289, 329-336(1991). The statistical significance of measured biochemical differencesbetween untreated and treated X-ALD cells can be determined by atwo-tailed Student's t-test.

Compounds of the invention were found to decrease the cellular contentof the VLCFA by approximately 60 percent in the X-ALD cells.

Example 32 Cystic Fibrosis Screening Assay

As described above, during its biosynthesis, CFTR is initiallysynthesized as a nascent polypeptide chain in the rough ER, with amolecular weight of around 120 kDa (B and A). It rapidly receives a coreglycosylation in the ER, giving it a molecular weight of around 140 kDa(Band B). As CFTR exits the ER and matures through the Golgi stacks, itsglycosylation is modified until it achieves a terminal matureglycosylation, affording it a molecular weight of around 170 kDa (BandC). The extent to which CFTR exits the ER and traverses the Golgi toreach the plasma membrane may be reflected in the ratio of Band B toBand C protein. CFTR is immunoprecipitated from control cells, and cellsexposed to test compounds. Both wt CFTR and AF508 CFTR expressing cellsare tested. Following lysis, CFTR are immunoprecipitated using variousCFTR antibodies. Immunoprecipitates are then subjected to in vitrophosphorylation using radioactive ATP and exogenous protein kinase A.Samples are subsequently solubilized and resolved by SDS-PAGE. Gels arethen dried and subject to autoradiography and phosphor image analysisfor quantitation of Bands B and C are determined on a BioRad personalfix image station.

Cell Culture

Chinese hamster ovary (CHO) cells stably expressing both wt and AF508CFTR were used in these assays. The cultures were grown on 100 mmplastic cell dishes in DMEM containing 10% foetal bovine serum (FBS) andkept at 5% CO₂/95% O₂ at 37° C. Cells were grown to confluence and used3-5 days post-plating. All test compounds were added to cells for 24hours prior to analysis.

Immunoprecipitation

Cells were treated with test compounds and CFTR immunoprecipitated asdescribed in Bradbury et al., Am. J. Physiol. 276, L659-668 (1999).Briefly, treated cells were lysed in buffer containing 1% TRITON X-100and various protease inhibitors. Soluble material was immunoprecipitatedusing both R domain and C-terminal monoclonal antibodies.Immunoprecipitated CFTR was then subject to in vitro phosphorylationusing camp-dependent PKA catalytic subunit and [γ-32P]ATP, followed byresolution on SDS-PAGE gels. After fixation, the gels were dried andprocessed for autoradiography and phosphor image analysis. Quantitationof B and C bands on a BioRad personal fix image analysis station.

It was found that compounds of the invention (at 100 μM) showed nosignificant changes in the levels of Bands B and C in treated cellsrelative to untreated cells. Based on the results obtained from usingthese test compounds, there was no gross effect of the test compounds onthe expression levels of wild type CFTR. Analysis of band C of ΔF508CFTR CHO cells showed that very little Band C was present in ΔF508 cellscompared to wild-type cells. Exposure of these cells to test compoundsat 100 μM for 24 hours at 37° C. did not affect the level of Band C CFTRin either wild-type or ΔF508 CFTR expressing cells. In contrast,analysis of Band B CFTR in ΔF508 cells showed that test compounds at 100μM resulted in a significant increase (about 6-7 fold) in the level of Band B compared to ΔF508 cells not exposed to the test compounds.

Example 33 Toxicity Assay

Test compounds of the invention were administered to three groups of 10mice at 100, 300, and 1,000 mg/kg. An additional group received vehicle(20% hydroxypropyl-β-cyclodextrin aqueous solution) at 10 mL/kg.Mortality/morbidity checks were made twice daily. Clinical observationswere recorded predose and/or postdose on Day 1, and daily thereafterthrough Day 8. Body weights were recorded on the day of dosing (Day 1)and on Day 8. Mice were euthanized by CO₂ asphyxiation and necropsied onDay 8 or upon death.

One test compound was tested so far and based on the results obtained,the no-observed toxicity level for this compound when administered toCD-1 mice as a single intraperitoneal does 100 mg/kg. Clinical signs oftoxicity were noted after dosing at 300 mg/kg with recovery within 24hours, while dosing at 1,000 mg/kg resulted in death (80% of animals) bythe end of Day 2.

Other Embodiments

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

1. A method of inhibiting histone deacetylase in cells comprisingcontacting the cells with an effective amount of a compound of formula(I):

wherein A is phenyl optionally substituted with alkyl, alkenyl, alkynyl,alkoxy, hydroxyl, or halo; Y¹ is a bond, or CH₂, and Y² is a bond, L isan unsaturated C₄ hydrocarbon chain with two double bonds, C₆hydrocarbon chain with three double bonds, or C₈ hydrocarbon chain withfour double bonds, said hydrocarbon chain being optionally substitutedwith C₁₋₄ alkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl; X¹ is O; and X² is NHOH; ora salt thereof; and determining whether the level of acetylated histonesin the treated cells is higher than in untreated cells under the sameconditions.